Award details

Fundamental connections between protein synthesis and carbohydrate metabolism: eIF4A regulation

Reference	BB/K005979/1
Principal Investigator / Supervisor	Professor Mark Peter Ashe
Co-Investigators / Co-Supervisors
Institution	The University of Manchester
Department	School of Biological Sciences
Funding type	Research
Value (£)	387,304
Status	Completed
Type	Research Grant
Start date	02/01/2013
End date	05/11/2016
Duration	46 months

Abstract

The identity and abundance of proteins shapes cellular traits and functions. Control of translation is therefore fundamental in numerous biological contexts. One such context is cellular stress, which commonly elicits a global translation inhibition to allow reprogramming of gene expression towards adaptive pathways. In the yeast S. cerevisiae glucose starvation leads to the most dramatic translation inhibition across various stress conditions. Previously, my lab have shown that glucose depletion inhibits translation initiation by a mechanism outside the known pathways of translation control. Our recent work reveals that glucose depletion elicits a rapid loss of eIF4A from the translation machinery and an accumulation of the 48S pre-initiation complex. Therefore, the affinity of eIF4A within the eIF4F complex must rapidly decrease leading to an accumulation of a stalled pre-initiation complex. This proposal will establish how the loss in eIF4A is brought about by investigating components of the eIF4F complex in terms of post-translational modifications, protein interactions and affinities under different conditions. The consequences of eIF4A loss from the translation machinery will also be compared using different methods to induce eIF4A loss. Finally, we will evaluate whether mRNAs that escape translation repression after glucose depletion rely upon eIF4A or on other helicases implicated in translation. Global translation control pathways are commonly conserved from yeast to humans, but often the cue that signals translation inhibition varies across different organisms. Careful mechansitic dissection in yeast of the novel pathway of translational control described in this proposal will provide a framework for future studies on potential conservation and phyisology in higher eukaryotes. If the pathway is not conserved in humans, there is an intriguing possibility that a targeted approach could be developed as a strategy to treat pathogenic fungal infections.

Summary

The information carried in the genes from living cells is decoded to produce chains of different amino acids called proteins that dictate the identity and function of that cell. Proteins represent one of the building blocks of all life, catalyzing most of the biochemical reactions as well as serving numerous structural and regulatory roles. An intermediate between the gene and the protein is the messenger RNA (mRNA). In eukaryotic cells (animals, plants and fungi), genes have been segregated into the nucleus away from the machinery involved in protein synthesis allowing independent regulation of mRNA and protein production. The 'translation' of the code contained in the mRNA is a complicated process that is highly similar across eukaryotic cells. All organisms need to be able to adapt to changes in their external environment. This adaptation involves changing the levels of individual proteins within cells. Hence, in response to stresses such as increases in temperature or starvation for certain nutrients, cells rapidly halt the process of protein synthesis to allow a switch to a stress specific protein production pathway. We are studying this regulation of translation using the simple eukaryote, brewer's yeast, as a model organism. Sugars are essential for life. Fundamentally, the breakdown of sugars provides the chemical energy that drives most other processes in living cells. The sugar, glucose, is used as the major source of energy in most living systems from bacteria to humans. For instance, as simple eukaryotic micro-organisms, yeast prefer to grow on glucose as a sugar source. One of the most dramatic decreases in protein production observed in response to stress in yeast occurs rapidly after the depletion of glucose from the growth media. We have recently discovered that a specific and critical translation factor dissociates from the translation machinery after glucose starvation. This factor is called the eukaryotic translation initiation factor 4A (eIF4A). eIF4A is an example of a class of proteins called helicases. These proteins can unwind DNA and RNA molecules, and eIF4A can unwind sections of the mRNA. This is thought to give the translation machinery access and promote the overall transit or 'scanning' of this machinery along the mRNA in search of an initiation site. Therefore in this proposal, we will study how the removal of glucose from yeast cells causes a loss of eIF4A from translating mRNAs. We seek to answer questions relating to what causes this reduction in the level of eIF4A associated with the translation machinery, how important this eIF4A loss is in the inhibition of protein production and whether mRNAs that continue to be translated are bound by eIF4A or other helicases. This project is important because it brings together two pathways that are fundamental to living systems these are the energy generating system and the protein production system. A detailed characterisation of this mechanism in brewer's yeast will facilitate the longer term investigation in other organisms. Should such a pathway of translation control be present in other organisms (possibly activated by different stresses) then it is likely to be very important for the physiology of that organism. Should the pathway prove to be fungal-specific then it might represent a new target for drug design to combat human pathogenic fungi.

Impact Summary

Who will benefit from this research? The impact from this project outside of academia can be divided into two areas, medical and industrial. In terms of medicine, translational control pathways play a role in a host of medically related topics, such as the defense against viral infection, iron homeostasis, severe diabetus mellitus, arsenite toxicity, oxidative stress and nutrient deprivation. This project may have specific impact as the translational target of glucose starvation in yeast is eIF4A and drugs such as patemine A, hippuristanol and silvestrol that target eIF4A are currently being evaluated as anti-cancer and anti-viral drugs. In addition, the Burkholderia pseudomallei pathogen has recently been shown to target eIF4A via the activity of the Burkholderia lethal factor 1 (BLF1) factor. Other drugs which inhibit translation initiation via effects on mTOR, such as Rapamycin (Sirolimus) are currently in use as a treatments for transplant rejection, whereas temsirolimus and everolimus are being evaluated as anti-cancer therapies. Furthermore, the discovery of human disease mutations (e.g. VWM and Wolcott-Rallison syndromes) in translation initiation factors and the kinases that regulate these factors highlights the extent to which translational control is an important clinical target. Should the regulation of translation of initiation by glucose starvation rely on residues/ proteins that are specific to fungi then this pathway could become a target for antifungal therapeutic strategies. Systemic infections with fungal pathogens such as Candida albicans and Aspergillus fumigatus are very difficult to treat and often fatal. Therefore it is possible that patients and practitioners from a wide range of medical disciplines could benefit from this project. In terms of industrial beneficiaries, any company using eukaryotic organisms for bioprocessing or synthetic biology will benefit from greater understanding of mechanisms that might globally down-regulate protein synthesis under specific growth or stress conditions. How will they benefit from this research? The results of the research proposed here will impact on the general understanding of the interplay between energy metabolism and protein synthesis in eukaryotes. This greater understanding could inform and improve treatments of diseases like cancer and viral infections. Moreover this work could lead to increased efficiency in bioprocessing applications. What will be done to ensure that they benefit from this research? Results will be disseminated through research seminars, presentations at conferences and publications in scientific journals. Funding is requested to attend national and international research conferences to allow the researchers to publicise this research. Resources generated from this project are likely to include next generation sequencing data, phopshoproteomics data, yeast strains and plasmids and will be made available to the scientific community upon request. Detailed protocols and primary data will be made freely available to academic collaborators. Manchester University has a good track record of encouraging public engagement. This includes regular open days to inform school children and the public about University research and tours of the research facilities at Manchester. This will allow the researchers to share their research findings with the wider public and to raise awareness of the importance of basic research. Manchester University maintains excellent links with the business sector, which will allow us to exploit any potential for collaboration with industry. This is managed by the faculty Business Development Team, who provide support and information for staff wishing to develop relationships with business.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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